1 Listeria monocytogenes (Lm) is a category B priority pathogen that causes outbreaks of 2 foodborne illness with a high incidence of morbidity and mortality. To achieve sterilizing 3 immunity against Lm, CD8+ T cells must recognize antigens bound to MHC-I proteins on the 4 surface of infected cells, an event that results in activation of the T cells and acquisition of 5 protective effector functions. Classical MHC-I proteins (MHC-Ia) have been studied for 6 decades, however, comparatively little is known about most of the non-classical MHC-I proteins 7 (MHC-Ib). We developed a MHC-Ia deficient mouse model of Lm infection to study the role of 8 MHC-Ib restricted T cells in the clearance of intracellular bacterial pathogens. Our central 9 hypothesis is that Lm-immune mice contain memory CD8+ T cells that recognize novel MHC-Ib 10 proteins, and that these T cells play a significant role in the clearance of secondary Lm infection. 11 In preliminary studies, we showed that CD8+ T cells that recognize antigen in the context of a 12 novel (not M3) MHC-Ib protein are activated during Lm infection. We have identified nine 13 murine MHC-Ib genes as likely candidates to express proteins that could serve as antigen 14 presenting molecules during infection. In this application, we propose to: 1) develop a panel of 15 human macrophage-like cells transfected with each of the nine candidate MHC-Ib genes and 2) 16 use the MHC-Ib transfectants to determine how many different MHC-Ib proteins are capable of 17 presenting antigen to T cells during Lm infection. These studies will help to define the role of 18 MHC-Ib restricted T cells in protective immune responses against Lm and may facilitate the 19 identification of new classes of antigens for all intracellular bacterial pathogens. Since most 20 MHC-Ib proteins are non-polymorphic, antigens that bind to MHC-Ib proteins are likely to be 21 recognized by most, if not all of the individuals in a given population. This makes MHC-Ib 22 antigens particularly attractive candidates for inclusion in vaccines designed to protect against 23 infection with intracellular bacterial pathogens. PUBLIC HEALTH RELEVANCE: One of the significant hurdles faced in trying to design vaccines to protect against infection with bacteria that can survive inside host cells is the identification of antigens that will be recognized by all individuals, regardless of their blood type (MHC haplotype). In this study, we will characterize a subset of T cells that recognize antigens derived from Listeria monocytogenes bound to MHC-Ib proteins, a class of proteins that is non- polymorphic (displays little variation among individuals) in both mice and humans. These studies have the potential to lead to new therapeutic strategies to protect against infections with a variety of intracellular bacteria, including prevalent human pathogens such as Mycobacteria tuberculosis and Chlamydia trachomatis.